Retinal derivatives and methods for the use thereof for the treatment of visual disorders

ABSTRACT

Compositions of and methods for using synthetic retinal derivatives as retinoid replacements and opsin agonists are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/042,308, filed Mar. 7, 2011, which claims the benefit of priority toU.S. patent application Ser. No. 11/629,875, now U.S. Pat. No.7,951,841, filed Feb. 12, 2008, under 35 U.S.C. §371(c) (1) as a U.S.national stage entry of International Patent Application No.PCT/US2005/021812, filed Jun. 20, 2005 under the Patent CooperationTreaty, and claims the benefit of priority to U.S. Provisional PatentApplication No. 60/580,889, filed Jun. 18, 2004, each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING GOVERNMENT INTEREST

This research was supported by United States Public Health Service GrantEY09339 from the National Eye Institute of the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

A diminished visual acuity or total loss of vision may result from anumber of eye diseases or disorders caused by dysfunction of tissues orstructures in the anterior segment of the eye and/or posterior segmentof the eye. Disease or disorders of the posterior segment of the eye ingeneral are retinal or choroidal vascular diseases or hereditarydiseases such as Leber Congenital Amaurosis. Age related maculardegeneration (AMD) is one of the specific diseases associated with theposterior portion of the eyeball and is the leading cause of blindnessamong older people. AMD results in damage to the macula, a smallcircular area in the center of the retina. Because the macula is thearea which enables one to discern small details and to read or drive,its deterioration may bring about diminished visual acuity and evenblindness. The retina contains two forms of light receiving cells, rodsand cones, that change light into electrical signals. The brain thenconverts these signals into the images. The macula is rich in conecells, which provides central vision. People with AMD sufferdeterioration of central vision but usually retain peripheral sight.

Slightly blurred or distorted vision is the most common early symptom ofAMD. Visual loss with dry AMD usually progresses slowly while visualloss with wet AMD proceeds more rapidly and may occur over days orweeks. Patients who have wet AMD in one eye are at increased risk ofdeveloping choroidal neo-vascularization (CNV) in the other eye. Themagnitude of the risk varies, depending on the appearance of the secondeye. The risk is greater in eyes with numerous large drusen, withabnormal pigment changes in the macula, and in patients with a historyof high blood pressure. Reactions that go on in the RPE lead tooxidative products leading to cell death and neovascularization. Thisexcess metabolism leads to the formation of drusen under the RPE.

Other eye diseases also affect photoreceptor function in the eye.Retinitis Pigmentosa represents disease caused by defects in manydifferent genes. They all have a final common pathway of night blindnessand peripheral vision loss that can lead to narrowing of the visualfield and eventual loss of all vision in many patients. The rodphotoreceptors are usually primarily affected and most of the genedefects leading to the disease occur in genes that are expressedpredominantly or only in the rod cells.

One autosomal dominant form of Retinitis Pigmentosa comprises an aminoacid substitution in opsin, a proline to histidine substitution at aminoacid 23. This defect compromises 10-20% of all Retinitis Pigmentosacases. This abnormal opsin protein forms a protein aggregate thateventually leads to cell death.

Leber Congenital Amaurosis is a very rare childhood condition thataffects children from birth or shortly thereafter. It affects both rodsand cones. There are a few different gene defects that have beenassociated with the disease. These include the genes encoding the RPE65and LRAT proteins. Both result in a person's inability to make11-cis-retinal in adequate quantities. In the RPE65-defectiveindividuals, retinyl esters build up in the retinal pigment epithelium(RPE). LRAT-defective individuals are unable to make esters andsubsequently secrete any excess retinoids.

Retinitis Punctata Albesciens is another form of Retinitis Pigmentosathat exhibits a shortage of 11-cis-retinal in the rods. Aging also leadsto the decrease in night vision and loss of contrast sensitivity due toa shorting of 11-cis-retinal. Excess unbound opsin is believed torandomly excite the visual transduction system. This can create noise inthe system, and thus more light and more contrast is necessary to seewell.

Congenital Stationary Night Blindness (CSNB) and Fundus Albipunctatusare a group of diseases that are manifested as night blindness, butthere is not a progressive loss of vision as in the RetinitisPigmentosa. Some forms of CSNB are due to a delay in the recycling of11-cis-retinal. Fundus Albipunctatus until recently was thought to be aspecial case of CSNB where the retinal appearance is abnormal withhundreds of small white dots appearing in the retina. It has been shownrecently that this is also a progressive disease although much slowerthan Retinitis Pigmentosa. It is caused by a gene defect that leads to adelay in the cycling of 11-cis-retinal.

Currently, there are few treatments for retinoid deficiency. Onetreatment, a combination of antioxidant vitamins and zinc, produces onlya small restorative effect. Thus, there is a need for compositions andmethods of restoring or stabilizing photoreceptor function andameliorating the effects of deficient levels of endogenous retinoids.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds and methods of using suchcompound to restore and/or stabilize photoreceptor function in avertebrate visual system. Synthetic retinal derivatives can beadministered to human or non-human vertebrate subjects to restore orstabilize photoreceptor function, and/or to ameliorate the effects of adeficiency in retinoid levels.

In one aspect, synthetic retinal derivatives are provided. The syntheticretinal derivative can be, for example, a derivative of Formula I, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV and/or XVI. Insome embodiments, the synthetic retinal derivatives is a retinyl ester,such as a 9-cis-retinyl ester or an 11-cis-retinyl ester. The estersubstituent can be, for example, a carboxylate radical of a C₃ to C₂₂polycarboxylic acid (polycarboxylate). For example, the substituent canbe succinate, citrate, ketoglutarate, fumarate, malate and oxaloacetate.In some embodiments, the ester substituent is not tartarate.

In some embodiments, the retinyl ester is a 9-cis-retinyl ester of a C₃to C₂₂ carboxylate. In other embodiments, the retinyl ester is a9-cis-retinyl ester of a C₃ to C₁₀ carboxylate. In some embodiments, theretinyl ester is an 11-cis-retinyl ester of a C₃ to C₂₂ carboxylate. Inother embodiments, the retinyl ester is an 11-cis-retinyl ester of a C₃to C₁₀ carboxylate.

Also provided are pharmaceutical compositions comprising the syntheticretinal derivative and a pharmaceutically acceptable vehicle. Thesynthetic retinal derivative can be, for example, a derivative ofFormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XVand/or XVI. In some embodiments, the synthetic retinal derivatives is aretinyl ester, such as a 9-cis-retinyl ester or an 11-cis-retinyl ester.The ester substituent can be, for example, a carboxylate radical of a C₃to C₂₂ polycarboxylic acid. The pharmaceutical composition can becompounded, for example, as an opthalmological composition in anopthalmologically acceptable vehicle for administration to the eyetopically or by intra-ocular injection.

In another aspect, a method of restoring photoreceptor function in amammal is provided. The method includes administering to a mammaliansubject having an endogenous retinoid deficiency an effective amount ofa synthetic retinal derivative, wherein the synthetic retinal derivativeis converted into a retinal capable of forming a functionalopsin/retinal complex. The synthetic retinal derivative can be, forexample, a 9-cis-retinyl ester, an 11-cis-retinyl ester, or acombination thereof. The ester substituent can be a carboxylate radicalof a C₁-C₁₀ monocarboxylic acid or a C₂ to C₂₂ polycarboxylic acid. Insome embodiments, synthetic retinal derivative is 9-cis-retinyl acetateor 11-cis-retinyl acetate. In other embodiments, the ester substituentcomprises a carboxylate radical of a polycarboxylic acid of C₃ to C₁₀.For example, the ester substituent can be succinate, citrate,ketoglutarate, fumarate, malate and oxaloacetate. The mammalian subjectcan be, for example, human or other mammal.

In another aspect, a method of ameliorating loss of photoreceptorfunction in a mammal is provided. The method includes administering aneffective amount of a synthetic retinal derivative to the vertebrateeye, wherein the synthetic retinal derivative is converted into aretinal capable of forming a functional opsin/retinal complex. Thesynthetic retinal derivative can be, for example, a 9-cis-retinyl ester,an 11-cis-retinyl ester, or a combination thereof. The ester substituentcan be a carboxylate radical of a C₁-C₁₀ monocarboxylic acid or a C₂ toC₂₂ polycarboxylic acid. In some embodiments, synthetic retinalderivative is 9-cis-retinyl acetate or 11-cis-retinyl acetate. In otherembodiments, the ester substituent comprises a carboxylate radical of apolycarboxylic acid of C₃ to C₁₀. For example, the ester substituent canbe succinate, citrate, ketoglutarate, fumarate, malate and oxaloacetate.The mammalian subject can be, for example, human or other mammal.

In an aspect, a method of restoring photoreceptor function in avertebrate eye is provided. The method can include administering to thevertebrate in need thereof having an endogenous retinoid deficiency aneffective amount of a synthetic retinal derivative in a pharmaceuticallyacceptable vehicle, wherein the synthetic retinal derivative isconverted into a retinal capable of forming a functional opsin/retinalcomplex. The synthetic retinal derivative can be, for example, aderivative of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII, XIV, XV and/or XVI. In some methods, if the synthetic retinalderivative is a 9-cis-retinyl ester comprising a monocarboxylic acidester substituent, it is a 9-cis-retinyl C₁ to C₁₀ ester. In somemethods, if the synthetic retinal derivative is an 11-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is an11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the synthetic retinal derivative can be administered toa vertebrate in need thereof. For example, the vertebrate can have, orbe predisposed to developing, an endogenous retinoid deficiencyassociated with Age-Related Macular Degeneration, Leber CongenitalAmaurosis, Retinitis Punctata Albesciens, Congenital Stationary NightBlindness, Fundus Albipunctatus, or other disease or conditionassociated with an endogenous retinoid deficiency.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In another aspect, a method of sparing the requirement for endogenousretinoid in a vertebrate eye is provided. The method can includeadministering to the eye a synthetic retinal derivative in apharmaceutically or ophthalmologically acceptable vehicle, wherein thesynthetic retinal derivative is converted into a retinal capable offorming a functional opsin/retinal complex. The synthetic retinalderivative can be, for example, a derivative of Formula I, II, III, IV,V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV and/or XVI. In somemethods, if the synthetic retinal derivative is a 9-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is a9-cis-retinyl C₁ to C₁₀ ester. In some methods, if the synthetic retinalderivative is an 11-cis-retinyl ester comprising a monocarboxylic acidester substituent, it is an 11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate;11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the synthetic retinal derivative can be administered toa vertebrate in need thereof. For example, the vertebrate can have, orbe predisposed to developing, an endogenous retinoid deficiencyassociated with Age-Related Macular Degeneration, Leber CongenitalAmaurosis, Retinitis Punctata Albesciens, Congenital Stationary NightBlindness, Fundus Albipunctatus, or other disease or conditionassociated with an endogenous retinoid deficiency.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In yet another aspect, a method of ameliorating loss of photoreceptorfunction in a vertebrate eye is provided. The method can includeprophylactically administering an effective amount of a syntheticretinal derivative in a pharmaceutically or ophthalmologicallyacceptable vehicle to the vertebrate eye, wherein the synthetic retinalderivative is converted into a retinal capable of forming a functionalopsin/retinal complex. The synthetic retinal derivative can be, forexample, a derivative of Formula I, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, XIII, XIV, XV and/or XVI. In some methods, if the syntheticretinal derivative is a 9-cis-retinyl ester comprising a monocarboxylicacid ester substituent, it is a 9-cis-retinyl C₁ to C₁₀ ester. In somemethods, if the synthetic retinal derivative is an 11-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is an11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In yet a further aspect, a method of selecting a treatment for a subjecthaving diminished visual capacity is provided. The method can includedetermining whether the subject has a deficient endogenous retinoidlevel, as compared with a standard subject; and administering to thesubject an effective amount of a synthetic retinal derivative in apharmaceutically acceptable vehicle (e.g., an ophthalmologicallyacceptable vehicle), wherein the synthetic retinal derivative isconverted into a retinal capable of forming a functional opsin/retinalcomplex. The synthetic retinal derivative can be, for example, aderivative of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII, XIV, XV and/or XVI. In some methods, if the synthetic retinalderivative is a 9-cis-retinyl ester comprising a monocarboxylic acidester substituent, it is a 9-cis-retinyl C₁ to C₁₀ ester. In somemethods, if the synthetic retinal derivative is an 11-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is an11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the endogenous retinoid is an 11-cis-retinyl ester. Insome methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In yet a further aspect, pharmaceutical compositions and oral dosageforms are provided. The compositions can include a synthetic retinalderivative in a pharmaceutically acceptable vehicle (e.g., anophthalmologically acceptable vehicle). The synthetic retinal derivativecan be, for example, a derivative of Formula I, II, III, IV, V, VI, VII,VIII, IX, X, XI, XII, XIII, XIV, XV and/or XVI. In some methods, if thesynthetic retinal derivative is a 9-cis-retinyl ester comprising amonocarboxylic acid ester substituent, it is a 9-cis-retinyl C₁ to C₁₀ester. In some methods, if the synthetic retinal derivative is an11-cis-retinyl ester comprising a monocarboxylic acid ester substituent,it is an 11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

The pharmaceutical composition can be, for example, an intraocularinjectable solution or a periocular injectable solution. The oral dosageform can be, for example, a pill, tablet, capsule, gel cap, or the like.

In yet another aspect, a method of treating Leber Congenital Amaurosisin a human subject is provided. The method generally includesadministering to a subject in need thereof an effective amount of asynthetic retinal derivative in a pharmaceutically or ophthalmologicallyacceptable vehicle. The synthetic retinal derivative can be, forexample, a derivative of Formula I, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, XIII, XIV, XV and/or XVI. In some methods, if the syntheticretinal derivative is a 9-cis-retinyl ester comprising a monocarboxylicacid ester substituent, it is a 9-cis-retinyl C₁ to C₁₀ ester. In somemethods, if the synthetic retinal derivative is an 11-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is an11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In another aspect, a method of treating Retinitis Punctata Albesciens,Congenital Stationary Night Blindness or Fundus Albipunctatus in a humansubject is provided. The method can include administering to the subjectin need thereof an effective amount of a synthetic retinal derivative ina pharmaceutically or ophthalmologically acceptable vehicle. The methodgenerally includes administering to a subject in need thereof aneffective amount of a synthetic retinal derivative in a pharmaceuticallyor ophthalmologically acceptable vehicle. The synthetic retinalderivative can be, for example, a derivative of Formula I, II, III, IV,V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV and/or XVI. In somemethods, if the synthetic retinal derivative is a 9-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is a9-cis-retinyl C₁ to C₁₀ ester. In some methods, if the synthetic retinalderivative is an 11-cis-retinyl ester comprising a monocarboxylic acidester substituent, it is an 11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is a 9-cis-retinylester, such as, for example, 9-cis-retinyl acetate, 9-cis-retinylsuccinate, 9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate,9-cis-retinyl fumarate, 9-cis-retinyl malate, 9-cis-retinyloxaloacetate, or the like. In some methods, the synthetic retinalderivative is an 11-cis-retinyl ester, such as, for example,11-cis-retinyl acetate, 11-cis-retinyl succinate, 11-cis-retinylcitrate, 11-cis-retinyl ketoglutarate, 11-cis-retinyl fumarate,11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or the like.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In yet another aspect, a method of treating Age-Related MacularDegeneration in a human subject is provided. The method can includeadministering to the subject in need thereof an effective amount of asynthetic retinal derivative in a pharmaceutically or ophthalmologicallyacceptable vehicle. The synthetic retinal derivative can be, forexample, a derivative of Formula I, II, III, N, V, VI, VII, VIII, IX, X,XI, XII, XIII, XIV, XV and/or XVI. In some methods, if the syntheticretinal derivative is a 9-cis-retinyl ester comprising a monocarboxylicacid ester substituent, it is a 9-cis-retinyl C₁ to C₁₀ ester. In somemethods, if the synthetic retinal derivative is an 11-cis-retinyl estercomprising a monocarboxylic acid ester substituent, it is an11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is converted into asynthetic retinal that binds to free opsin in the eye. In some methods,the synthetic retinal derivative is a 9-cis-retinyl ester, such as, forexample, 9-cis-retinyl acetate, 9-cis-retinyl succinate, 9-cis-retinylcitrate, 9-cis-retinyl ketoglutarate, 9-cis-retinyl fumarate,9-cis-retinyl malate, 9-cis-retinyl oxaloacetate, or the like. In somemethods, the synthetic retinal derivative is an 11-cis-retinyl ester,such as, for example, 11-cis-retinyl acetate, 11-cis-retinyl succinate,11-cis-retinyl citrate, 11-cis-retinyl ketoglutarate, 11-cis-retinylfumarate, 11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or thelike.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

In yet a further aspect, a method of treating or preventing loss ofnight vision or contrast sensitivity in an aging human subject isprovided. The method can include administering to the subject in needthereof an effective amount of a synthetic retinal derivative in apharmaceutically or ophthalmologically acceptable vehicle. The syntheticretinal derivative can be, for example, a derivative of Formula I, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV and/or XVI. Insome methods, if the synthetic retinal derivative is a 9-cis-retinylester comprising a monocarboxylic acid ester substituent, it is a9-cis-retinyl C₁ to C₁₀ ester. In some methods, if the synthetic retinalderivative is an 11-cis-retinyl ester comprising a monocarboxylic acidester substituent, it is an 11-cis-retinyl C₁ to C₁₀ ester.

In some methods, the synthetic retinal derivative is converted into asynthetic retinal that binds to free opsin in the eye. In some methods,the synthetic retinal derivative is a 9-cis-retinyl ester, such as, forexample, 9-cis-retinyl acetate, 9-cis-retinyl succinate, 9-cis-retinylcitrate, 9-cis-retinyl ketoglutarate, 9-cis-retinyl fumarate,9-cis-retinyl malate, 9-cis-retinyl oxaloacetate, or the like. In somemethods, the synthetic retinal derivative is an 11-cis-retinyl ester,such as, for example, 11-cis-retinyl acetate, 11-cis-retinyl succinate,11-cis-retinyl citrate, 11-cis-retinyl ketoglutarate, 11-cis-retinylfumarate, 11-cis-retinyl malate, 11-cis-retinyl oxaloacetate, or thelike.

In some methods, the synthetic retinoid derivative can be administeredlocally, such as by eye drops, intraocular injection, periocularinjection or the like. In other methods, the synthetic retinalderivative can be orally administered to the vertebrate. In somemethods, the vertebrate is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HPLC chromatogram showing retinoid elution in treated andcontrol mice eye and liver tissue. A. Eyes from dark adapted LRAT−/−mouse. B. Eyes from dark-adapted LRAT−/− mouse gavaged with 5 mgall-trans-retinyl palmitate 2 days prior. C. Eyes from dark-adaptedLRAT−/− mouse gavaged with 5 mg all-trans-retinyl acetate 2 days prior.D. Eyes from dark-adapted LRAT−/− mouse gavaged with 6.5 mg9-cis-retinyl acetate 3 days prior. E. Liver tissue from dark adaptedLRAT−/− mouse. F. Liver tissue from dark-adapted LRAT−/− mouse gavagedwith 5 mg all-trans-retinyl palmitate 2 days prior. G. Liver tissue fromdark-adapted LRAT−/− mouse gavaged with 5 mg all-trans-retinyl acetate 2days prior. H. Liver tissue from dark-adapted LRAT−/− mouse gavaged with6.5 mg 9-cis-retinyl acetate 3 days prior.

FIG. 2. Eye 9-cis-retinal oximes and 9-cis-retinol time course, 20 μMgavage.

FIG. 3. UV isomerization of all-trans-retinyl acetate to 9-cis-retinylacetate.

FIG. 4. HPLC separation of 13-cis-retinyl acetate (1), 9-cis-retinylacetate (2), and all-trans-retinyl acetate (3).

FIG. 5. Levels of 9-cis-retinal oximes in the eyes of Lrat−/− mice aftera single or multiple dose of 9-cis-R-Acetate. (a) The level of 9-cis-RALin Lrat−/− mouse eyes after a varying dose of 9-cis-R-Ac. (b) The levelof 9-cis-RAL in Lrat−/− mouse eyes after a varying size and number ofdoses of 9-cis-R-Ac.

FIG. 6. Chromophore levels (as 9-cis-retinal oximes) in the eyes ofLrat−/− mice after administration of all-trans-retinoid isomers or9-cis-retinyl succinate). The structures of the all-trans-retinoidisomers and 9-cis-retinyl succinate are also shown.

FIG. 7. A comparison of the chromophore levels (as 9-cis-retinal oximes)in the eyes of Lrat−/− mice after administration of 9-cis-retinal or9-cis-retinyl acetate at low and high doses.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides synthetical retinal derivatives andmethods of using such derivatives to restore or stabilize photoreceptorfunction in a vertebrate visual system. The synthetic retinal derivativeis a derivative of 9-cis-retinal or 11-cis-retinal in which thealdehydic group in the polyene chain is modified. The synthetic retinalderivative can be converted directly or indirectly into a retinal or asynthetic retinal analog. Thus, in some aspects, the compounds accordingto the present invention can be described as a pro-drug, which uponmetabolic transformation is converted into 9-cis-retinal, 11-cis-retinalor a synthetic retinal analog thereof. Metabolic transformation canoccur, for example by acid hydrolysis, esterase activity,acetyltransferase activity, dehydrogenase activity, or the like.

The synthetic retinal derivative can be a retinoid replacement,supplementing the levels of endogenous retinoid. In some embodiments,the synthetic retinal can bind to opsin, and function as an opsinagonist. As used herein, the term “agonist” refers to a syntheticretinal that binds to opsin and facilitates the ability of anopsin/synthetic retinal complex to respond to light. As an opsinagonist, a synthetic retinal can spare the requirement for endogenousretinoid (e.g., 11-cis-retinal). A synthetic retinal also can restore orimprove function (e.g., photoreception) to opsin by binding to opsin andforming a functional opsin/synthetic retinal complex, whereby theopsin/synthetic retinal complex can respond to photons when part of arod or cone membrane.

Synthetic retinal derivatives can be administered to restore orstabilize photoreceptor function, and/or to ameliorate the effects of adeficiency in retinoid levels. Photoreceptor function can be restored orstabilized, for example, by providing a synthetic retinal derivative asan 11-cis-retinoid replacement and/or an opsin agonist. The syntheticretinal derivative also can ameliorate the effects of a retinoiddeficiency on a vertebrate visual system. The synthetic retinalderivative can be administered prophylactically or therapeutically to avertebrate. Suitable vertebrates include, for example, human andnon-human vertebrates. Suitable non-human vertebrates include, forexample, mammals, such as dogs (canine), cats (feline), horses (equine)and other domesticated animals.

In one aspect, synthetic retinal derivatives are provided. The syntheticretinal derivatives are derivatives of 9-cis-retinal or 11-cis-retinalin which the aldehydic group in the polyene chain is converted to anester, ether, alcohol, hemi-acetal, acetal, oxime, as further describedherein. Such synthetic retinal derivatives include 9-cis-retinyl esters,9-cis-retinyl ethers, 9-cis-retinol, 9-cis-retinal oximes, 9-cis-retinylacetals, 9-cis-retinyl hemiacetals, 11-cis-retinyl esters,11-cis-retinyl ethers, 11-cis-retinol, 11-cis-retinyl oximes,11-cis-retinyl acetals and 11-cis-retinyl hemiacetals, as furtherdescribed herein. The synthetic retinal derivative can be metabolized torelease a natural or synthetic retinal, such as for example,9-cis-retinal, 11-cis-retinal or a synthetic retinal analog thereof,such as those described herein or in co-pending InternationalApplication No. PCT/US04/07937, filed Mar. 15, 2004, (Attorney DocketNo. 016336-002010PC) (the disclosure of which is incorporated byreference herein).

In one aspect, the synthetic retinal derivative is a retinyl ester. Insome embodiments, the retinyl ester is a 9-cis-retinyl ester or an11-cis-retinyl ester having a. The ester substituent can be, forexample, a carboxylic acid, such as a mono- or polycarboxylic acid. Asused herein, a “polycarboxylic acid” is a di-, tri- or higher ordercarboxylic acid. In some embodiments, the carboxylic acid is a C₁-C₂₂,C₂-C₂₂, C₃-C₂₂, C₁-C₁₀, C₂-C₁₀, C₃-C₁₀, C₄-C₁₀, C₄-C₈, C₄-C₆ or C₄monocarboxylic acid, or polycarboxylic acid.

Suitable carboxylic acid groups include, for example, acetic acid,propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid,pelargonic acid, capric acid, lauric acid, oleic acid, stearic acid,palmitic acid, myristic acid or linoleic acid. The carboxylic acid alsocan be, for example, oxalic acid (ethanedioic acid), malonic acid(propanedioic acid), succinic acid (butanedioic), fumaric acid(butenedioic acid), malic acid (2-hydroxybutenedioic acid), glutaricacid (pentanedioic acid), adipic acid (hexanedioic acid), pimelic acid(heptanedioic), suberic acid (octanedioic), azelaic acid (nonanedioicacid), sebacic acid (decanedioic acid), citric acid, oxaloacetic acid,ketoglutaratic acid, or the like.

In an exemplary embodiment, the retinyl ester is a 9-cis-retinyl esteror an 11-cis-retinyl ester including a C₃-C₁₀ polycarboxylic acidsubstituent. (In this context, the terms “substituent” or “group” referto a radical covalently linked to the terminal oxygen in the polyenechain.) In another exemplary embodiment, the retinyl ester is a9-cis-retinyl ester or an 11-cis-retinyl ester including a C₂-C₂₂ orC₃-C₂₂ polycarboxylic acid substituent. The polycarboxylic acidsubstituent can be, for example, succinate, citrate, ketoglutarate,fumarate, malate or oxaloacetate. In another exemplary embodiment, theretinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl esterincluding a C₃-C₂₂ di-carboxylic acid (di-acid) substituent. In someembodiments, the polycarboxylic acid is not 9-cis-retinyl tartarate or11-cis-retinyl tartarate. In some embodiments, the retinyl ester is nota naturally occurring retinyl ester normally found in the eye. In someembodiments, the retinyl ester is an isolated retinyl ester. As usedherein, “isolated” refers to a molecule that exists apart from itsnative environment and is therefore not a product of nature. An isolatedmolecule may exist in a purified form or may exist in a non-nativeenvironment.

In another aspect, the retinal derivative can be a 9-cis-retinyl esteror ether of the following formula I:

In some embodiments, A is CH₂OR, where R can be an aldehydic group, toform a retinyl ester. A suitable aldehydic group is a C₁ to C₂₄ straightchain or branched aldehydic group. The aldehydic group also can be a C₁to C₁₄ straight chain or branched aldehydic group. The aldehydic groupcan be a C₁ to C₁₂ straight chain or branched aldehydic group, such as,for example, acetaldehyde, propionaldehyde, butyraldehyde,valeraldehyde, hexanal, heptanal, octanal, nonanal, decanal, undecanal,dodecanal. R can be a C₁ to C₁₀ straight chain or branched aldehydicgroup, a C₁ to C₈ straight chain or branched aldehydic group or a C₁ toC₆ straight chain or branched aldehydic group.

R further can be a carboxylate group of a dicarboxylic acid or othercarboxylic acid (e.g., a hydroxyl acid) to form a retinyl ester (some ofwhich are also referred to as retinoyl esters). The carboxylic acid canbe, for example, oxalic acid (ethanedioic acid), malonic acid(propanedioic acid), succinic acid (butanedioic), fumaric acid(butenedioic acid), malic acid (2-hydroxybutenedioic acid), glutaricacid (pentanedioic acid), adipic acid (hexanedioic acid), pimelic acid(heptanedioic), suberic acid (octanedioic), azelaic acid (nonanedioicacid), sebacic acid (decanedioic acid), citric acid, oxaloacetic acid,ketoglutaratic acid, or the like.

R can also be an alkane group, to form a retinyl alkane ether. Suitablealkane groups include, for example, C₁ to C₂₄ straight chain or branchedalkyls, such as, for example, methane, ethane, butane, isobutane,pentane, isopentane, hexane, heptane, octane or the like. For example,the alkane group can be a linear, iso-, sec-, tert- or other branchedlower alkyl ranging from C₁ to C₆. The alkane group also can be alinear, iso-, sec-, tert- or other branched medium chain length alkylranging from C₈ to C₁₄. The alkane group also can be a linear, iso-,sec-, tert- or other branched long chain length alkyl ranging from C₁₆to C₂₄.

R further can be an alcohol group, to form a retinyl alcohol ether.Suitable alcohol groups can be linear, iso-, sec-, tert- or otherbranched lower alcohols ranging from C₁ to C₆, linear, iso-, sec-, tert-or other branched medium chain length alcohols ranging from C₈ to C₁₄,or linear, iso-, sec-, tert- or other branched long chain length alkylranging from C₁₆ to C₂₄. The alcohol group can be, for example,methanol, ethanol, butanol, isobutanol, pentanol, hexanol, heptanol,octanol, or the like

R also can be a carboxylic acid, to form a retinyl carboxylic acidether. Suitable alcohol groups can be linear, iso-, sec-, tert- or otherbranched lower carboxylic acids ranging from C₁ to C₆, linear, iso-,sec-, tert- or other branched medium chain length carboxylic acidsranging from C₈ to C₁₄, or linear, iso-, sec-, tert- or other branchedlong chain length carboxylic acids ranging from C₁₆ to C₂₄. Suitablecarboxylic acid groups include, for example, acetic acid, propionicacid, butyric acid, valeric acid, caproic acid, caprylic acid,pelargonic acid, capric acid, lauric acid, oleic acid, stearic acid,palmitic acid, myristic acid, linoleic acid, succinic acid, fumaric acidor the like.

The retinyl derivative can be a retinyl hemiacetal, where A is CH(OH)OR.R can be any of the R groups set forth above in Formula I. R istypically a lower alkane, such as a methyl or ethyl group, or a C₁ to C₇saturated and unsaturated, cyclic or acyclic alkane, with or withouthetero atoms, as described herein.

The retinyl derivative can be a retinyl acetal, where A isCH(OR_(a))OR_(b). Each of R_(a) and R_(b) can be independently selectedfrom any of the R groups set forth above in Formula I. R_(a) and R_(b)are typically a C₁ to C₇ saturated and unsaturated, cyclic or acyclicalkane, with or without hetero atoms, as described herein.

The retinyl derivative also can be a retinyl oxime, where A is CH:NOH orCH:NOR. R can be any of the R groups set forth above in Formula I. R istypically a hydrogen, or an alkane.

Examples of suitable synthetic retinal derivatives include, for example,9-cis-retinyl acetate, 9-cis-retinyl formate, 9-cis-retinyl succinate,9-cis-retinyl citrate, 9-cis-retinyl ketoglutarate, 9-cis-retinylfumarate, 9-cis-retinyl malate, 9-cis-retinyl oxaloacetate,9-cis-retinal oxime, 9-cis-retinal O-methyl oximes, 9-cis-retinalO-ethyl oximes, and 9-cis-retinal methyl acetals and hemi acetals,9-cis-retinyl methyl ether, 9-cis-retinyl ethyl ether, and 9-cis-retinylphenyl ether.

In a related aspect, the retinal derivative can be an 11-cis-retinylester or ether of the following formula II:

A can be any of the groups set forth above in Formula I.

Examples of suitable synthetic retinal derivatives include, for example,11-cis-retinyl acetate, 11-cis-retinyl formate, 11-cis-retinylsuccinate, 11-cis-retinyl citrate, 11-cis-retinyl ketoglutarate,11-cis-retinyl fumarate, 11-cis-retinyl malate, 11-cis-retinal oxime,11-cis-retinal O-methyl oxime, 11-cis-retinal O-ethyl oximes and11-cis-retinal methyl acetals and hemi acetals, 11-cis-retinyl methylether, 11-cis-retinyl ethyl ether.

In additional aspects, the synthetic retinal derivatives can be, forexample, a derivative of a 9-cis-retinyl ester, a 9-cis-retinyl ether,an 11-cis-retinyl ester or an 11-cis-retinyl ethers such as, forexample, an acyclic retinyl ester or ethers, a retinyl ester or etherwith a modified polyene chain length, such as a trienoic or tetraenoicretinyl ester or ether; a retinyl ester or ether with a substitutedpolyene chain, such as alkyl, halogen or heteratom-substituted polyenechains; a retinyl ester or ether with a modified polyene chain, such asa trans- or cis-locked polyene chain, or with, for example, allene oralkyne modifications; and a retinyl ester or ether with a ringmodification(s), such as heterocyclic, heteroaromatic or substitutedcycloalkane or cycloalkene rings.

The synthetic retinal derivative can be a retinyl ester or ether of thefollowing formula III:

A can be any of the groups set forth above for formula (I). R₁ and R₂can be independently selected from linear, iso-, sec-, tert- and otherbranched alkyl groups as well as substituted alkyl groups, substitutedbranched alkyl, hydroxyl, hydroalkyl, amine, amide, or the like. R₁ andR₂ can independently be lower alkyl, which means straight or branchedalkyl with 1-6 carbon atom(s) such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl, hexyl, or the like. Suitablesubstituted alkyls and substituted branch alkyls include, for example,alkyls, branched alkyls and cyclo-alkyls substituted with oxygen,hydroxyl, nitrogen, amide, amine, halogen, heteroatom or other groups.Suitable heteroatoms include, for example, sulfur, silicon, and fluoro-or bromo-substitutions.

R₁ or R₂ also can be a cyclo-alkyl such as, for example, hexane,cyclohexene, benzene as well as a substituted cyclo-alkyl. Suitablesubstituted cyclo-alkyls include, for example, cyclo-alkyls substitutedwith oxygen, hydroxyl, nitrogen, amide, amine, halogen, heteroatomand/or other groups. Suitable heteroatoms include, for example, sulfur,silicon, and fluoro- or bromo-substitutions.

The synthetic retinal derivative also can have a modified polyene chainlength, such as the following formula IV:

A can be any of the groups set forth above for formula (I). The polyenechain length can be extended by 1, 2, or 3 alkyl, alkene or alkylenegroups. According to formula (IV), each n and n₁ can be independentlyselected from 1, 2, or 3 alkyl, alkene or alkylene groups, with theproviso that the sum of the n and n₁ is at least 1.

The synthetic retinal derivative also can have a substituted polyenechain of the following formula V:

A can be any of the groups set forth above for formula (I). Each of R₁to R₈ can be independently selected from hydrogen, alkyl, branchedalkyl, cyclo-alkyl, halogen, a heteratom, or the like. Suitable alkylsinclude, for example, methyl, ethyl, propyl, substituted alkyl (e.g.,alkyl with hydroxyl, hydroalkyl, amine, amide) or the like. Suitablebranched alkyls can be, for example, isopropyl, isobutyl, substitutedbranched alkyl, or the like. Suitable cyclo-alkyls can include, forexample, cyclohexane, cycloheptane, and other cyclic alkanes as well assubstituted cyclic alkanes such as substituted cyclohexane orsubstituted cycloheptane. Suitable halogens include, for example,bromine, chlorine, fluorine, or the like. Suitable heteroatoms include,for example, sulfur, silicon, and fluoro- or bromo-substitutions.Suitable substituted alkyls, substituted branch alkyls and substitutedcyclo-alkyls include, for example, alkyls, branched alkyls andcyclo-alkyls substituted with oxygen, hydroxyl, nitrogen, amide, amine,halogen, heteroatom or other groups.

For example, the synthetic retinal derivative can be selected from thefollowing: a 9-ethyl-11-cis-retinyl ester, ether, oxime, acetal orhemiacetal; a 7-methyl-11-cis-retinyl ester, ether, oxime, acetal orhemiacetal; a 13-desmethyl-11-cis-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-Cl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-F-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-Cl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12-Cl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-12-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-10-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-F-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-Cl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-12-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-F-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; an 11-cis-14-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14-F-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14-methyl-retinyl ester, ether, oxime, acetal orhemiacetal; a 9-cis-14-ethyl-retinyl ester, ether, oxime, acetal orhemiacetal; or the like.

The synthetic retinal derivative further can have a modified ringstructure. Suitable examples include, for example, derivativescontaining ring modifications, aromatic analogs and heteroaromaticanalogs of the following formulae VI, VII and VIII, respectively:

A can be any of the groups set forth above for formula (I). Each of R₁to R₆, as applicable, can be independently selected from hydrogen,alkyl, substituted alkyl, hydroxyl, hydroalkyl, amine, amid; halogen, aheteratom, or the like. Suitable alkyls include, for example, methyl,ethyl, propyl, isopropyl, butyl, isobutyl or the like. Suitable halogensinclude, for example, bromine, chlorine, fluorine, or the like. Suitableheteroatoms include, for example, sulfur, silicon, or nitrogen. Informulae VII, X can be, for example, sulfur, silicon, nitrogen, fluoro-or bromo-substitutions. Similarly, 9-cis-synthetic retinal derivativescontaining ring modifications, aromatic analogs and heteroaromaticanalogs of those shown in formulae VI, VII and VIII are contemplated.

The synthetic retinal derivative also can have a modified polyene chain.Suitable derivatives include, for example, those with a trans/cis lockedconfiguration, 6s-locked analogs, as well as modified allene, alkene,alkyne or alkylene groups in the polyene chain. In one example, thederivative is an 11-cis-locked analog of the following formula IX:

A can be any of the groups set forth above for formula (I). R₃ can be,for example, hydrogen, methyl or other lower alkane or branch alkane. ncan be 0 to 4. m plus 1 equals 1, 2 or 3.

In one embodiment, the synthetic retinal derivative can be an11-cis-locked analog of the following formula X:

n can be 1 to 4. A can be any of the groups set forth above for formula(I).

The synthetic retinal derivative is a 9,11,13-tri-cis-7-ring retinylester or ether, an 11,13-di-cis-7-ring retinyl ester or ether, an11-cis-7-ring retinyl ester or ether or a 9,11-di-cis-7-ring retinylester or ether.

In another example, the synthetic retinal derivative is a 6s-lockedanalog of formula XI. A can be any of the groups set forth above forformula (I). R₁ and R₂ can be independently selected from hydrogen,methyl and other lower alkyl and substituted lower alkyl. R₃ can beindependently selected from an alkene group at either of the indicatedpositions.

The synthetic retinal derivative can be a 9-cis-ring-fused derivative,such as, for example, those shown in formulae XII-XIV. A can be any ofthe groups set forth above for formula (I).

The synthetic retinal derivative also can be of the following formula XVor XVI.

A can be any of the groups set forth above for formula (I). Each of R₂to R₅, R₇ to R₁₄, R₁₆ and R₁₇ can be absent or independently selectedfrom hydrogen, alkyl, branched alkyl, halogen, hydroxyl, hydroalkyl,amine, amide, a heteratom, or the like. Suitable alkyls include, forexample, methyl, ethyl, propyl, substituted alkyl (e.g., alkyl withhydroxyl, hydroalkyl, amine, amide), or the like. Suitable branchedalkyl can be, for example, isopropyl, isobutyl, substituted branchedalkyl, or the like. Suitable halogens include, for example, bromine,chlorine, fluorine, or the like. Suitable heteroatoms include, forexample, sulfur, silicon, and fluoro- or bromo-substitutions. Suitablesubstituted alkyls and substituted branch alkyls include, for example,alkyls and branched alkyls substituted with oxygen, hydroxyl, nitrogen,amide, amine, halogen, heteroatom or other groups.

Each of n and n₁ can be independently selected from 1, 2, or 3 alkyl,alkene or alkylene groups, with the proviso that the sum of the n and n₁is at least 1. In addition, R₃-R₄ and/or R₂-R₁₆ can comprise an alkenegroup in the cyclic carbon ring, in which case R₁₇ is absent. R₁₀ andR₁₃ together can form a cyclo-alkyl, such as a five, six, seven or eightmember cyclo-alkyl or substituted cyclo-alkyl, such as, for example,those shown in Formulae IX, X, XII, XIII and XIV.

Methods of making synthetic retinals and derivatives are disclosed in,for example, the following references: Anal. Biochem. 272:232-42 (1999);Angew. Chem. 36:2089-93 (1997); Biochemistry 14:3933-41 (1975);Biochemistry 21:384-93 (1982); Biochemistry 28:2732-39 (1989);Biochemistry 33:408-16 (1994); Biochemistry 35:6257-62 (1996);Bioorganic Chemistry 27:372-82 (1999); Biophys. Chem. 56:31-39 (1995);Biophys. J. 56:1259-65 (1989); Biophys. J. 83:3460-69 (2002); Chemistry7:4198-204 (2001); Chemistry (Europe) 5:1172-75 (1999); FEBS 158:1(1983); J. Am. Chem. Soc. 104:3214-16 (1982); J. Am. Chem. Soc.108:6077-78 (1986); J. Am. Chem. Soc. 109:6163 (1987); J. Am. Chem. Soc.112:7779-82 (1990); J. Am. Chem. Soc. 119:5758-59 (1997); J. Am. Chem.Soc. 121:5803-04 (1999); J. American Chem. Soc. 123:10024-29 (2001); J.American Chem. Soc. 124:7294-302 (2002); J. Biol. Chem. 276:26148-53(2001); J. Biol. Chem. 277:42315-24 (2004); J. Chem. Soc.-Perkin T.1:1773-77 (1997); J. Chem. Soc.-Perkin T 1:2430-39 (2001); J. Org. Chem.49:649-52 (1984); J. Org. Chem. 58:3533-37 (1993); J, Physical ChemistryB 102:2787-806 (1998); Lipids 8:558-65; Photochem. Photobiol. 13:259-83(1986); Photochem. Photobiol. 44:803-07 (1986); Photochem. Photobiol.54:969-76 (1991); Photochem. Photobiol. 60:64-68 (1994); Photochem.Photobiol. 65:1047-55 (1991); Photochem. Photobiol. 70:111-15 (2002);Photochem. Photobiol. 76:606-615 (2002); Proc. Natl. Acad. Sci. USA88:9412-16 (1991); Proc. Natl Acad. Sci. USA 90:4072-76 (1993); Proc.Natl. Acad. Sci, USA 94:13442-47 (1997); and Proc. R. Soc. Lond. SeriesB, Biol. Sci. 233 (1270): 55-76 1988) (the disclosures of which areincorporated by reference herein).

Retinyl esters can be formed by methods known in the art such as, forexample, by acid-catalyzed esterification of a retinol with a carboxylicacid, by reaction of an acyl halide with a retinol, bytransesterification of a retinyl ester with a carboxylic acid, byreaction of a primary halide with a carboxylate salt of a retinoic acid,by acid-catalyzed reaction of an anhydride with a retinol, or the like.In an example, retinyl esters can be formed by acid-catalyzedesterification of a retinol with a carboxylic acid, such as, aceticacid, propionic acid, butyric acid, valeric acid, caproic acid, caprylicacid, pelargonic acid, capric acid, lauric acid, oleic acid, stearaticacid, palmitic acid, myristic acid, linoleic acid, succinic acid,fumaric acid or the like. In another example, retinyl esters can beformed by reaction of an acyl halide with a retinol (see, e.g., VanHooser et al., Proc. Natl. Acad. Sci. USA, 97:8623-28 (2000)). Suitableacyl halides include, for example, acetyl chloride, palmitoyl chloride,or the like.

Retinyl ethers can be formed by methods known in the art, such as forexample, reaction of a retinol with a primary alkyl halide.

Trans-retinoids can be isomerized to cis-retinoids by exposure to UVlight. For example, all-trans-retinal, all-trans-retinol,all-trans-retinyl ester or all-trans-retinoic acid can be isomerized to9-cis-retinal, 9-cis-retinol, 9-cis-retinyl ester or 9-cis-retinoicacid, respectively. trans-Retinoids can be isomerized to 9-cis-retinoidsby, for example, exposure to a UV light having a wavelength of about 365nm, and substantially free of shorter wavelengths that cause degradationof cis-retinoids, as further described herein.

Retinyl acetals and hemiacetals can be prepared, for example, bytreatment of 9-cis- and 11-cis-retinals with alcohols in the presence ofacid catalysts. Water formed during reaction is removed, for example byAl₂O₃ of a molecular sieve.

Retinyl oximes can be prepared, for example, by reaction of a retinalwith hydroxylamine, O-methyl- or O-ethylhydroxylamine, or the like.

For a specific opsin protein, a suitable synthetic retinal derivativescan be identified, for example, by an expression system expressing theopsin protein. Suitable animal models include, for example, RPE65−/− orLRAT −/− mice (see, e.g., Van Hooser et al., J. Biol. Chem. 277:19173-82(2002); Baehr et al., Vision Res. 43:2957-58 (2003); Batten et al., J.Biol. Chem. 279:10422-32 (2004); Kuksa et al., Vision Res. 43:2959-81(2003); Thompson et al., Dev. Ophthalmol. 37:141-54 (2003)). Othersuitable non-human animal models further include other mouse, rat orprimate systems. Such animal models can be prepared, for example, bypromoting homologous recombination between a nucleic acid encoding anopsin in its chromosome and an exogenous nucleic acid encoding a mutantopsin. In one aspect, homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing anopsin gene, such that homologous recombination occurs, followed byinjecting the ES cells into a blastocyst, and implanting the blastocystinto a foster mother, followed by the birth of the chimeric animal (see,e.g., Capecchi, Science 244:1288-92 (1989)). The chimeric animal can bebred to produce additional transgenic animals.

Suitable expression systems also can include, for example, in vitro orin vivo systems. Suitable in vitro systems include for example, coupledtranscription-translation systems. Suitable in vivo systems include, forexample, cells expressing an opsin protein. For example, cells of avertebrate visual system can be adapted for culture in vitro, orrecombinant cell lines expressing an opsin protein can be used. The celllines are typically stable cell lines expressing the opsin protein. Asynthetic retinal or synthetic retinal derivative can be added to thecell culture media, and the cells cultured for a suitable period of timeto allow the production of opsin/rhodopsin. Opsin and/or rhodopsin canbe isolated (e.g., by immunoaffinity). Isolated protein samples areexamined to determine the amount of pigment formed, and absorbancemaxima. Methods of introducing nucleic acids into vertebrate cells aredisclosed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press (Cold SpringHarbor, N.Y., 2001).

Recombinant cell lines expressing opsin protein can be prepared by, forexample, introducing an expression construct encoding an opsin proteininto a suitable cell line. The expression construct typically includes apromoter operably linked to a nucleic acid encoding an opsin protein,and optionally a termination signal(s). Nucleic acids encoding opsin canbe obtained, for example, by using information from a database (e.g., agenomic or cDNA library), by polymerase chain reaction, or the like. Forexample opsin-encoding nucleic acids can be obtained by hybridization.(See generally Sambrook et al. (supra).) An opsin encoding nucleic acidcan be obtained by hybridization under conditions of low, medium or highstringency.

Opsin-encoding nucleic acids can be obtained under conditions of highstringency hybridization. By way of example, and not limitation,procedures using conditions of high stringency are as follows:Prehybridization of filters containing DNA is carried out for 8 hours toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 hours at 65°C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 65° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP,0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at50° C. for 45 minutes before autoradiography. Other conditions of highstringency which can be used are well known in the art. (See generallySambrook et al. (supra).)

The expression construct can optionally include one or more origins ofreplication and/or selectable marker(s) (e.g., an antibiotic resistancegene). Suitable selectable markers include, for example, thoseconferring resistance to ampicillin, tetracycline, neomycin, G418, andthe like. Suitable cell lines include, for example, HEK293 cells,T-REx™-293 cells, CHO cells and other cells or cell lines.

The UV-visible spectra of rhodopsin (comprising opsin and a syntheticretinal) can be monitored to determine whether the synthetic retinal hasformed a Schiff's base with the opsin protein. For example,acid-denatured, purified protein can be analyzed to determine whether anabsorbance maxima of approximately 490 nm is present, providing evidencethat the synthetic retinal derivative forms a Schiff's base with theopsin protein. Hydroxylamine treatment can be used to confirm theSchiff's base is sequestered from the external environment.

Suitable synthetic retinal derivatives also can be selected by molecularmodeling of rhodopsin. The coordinates for rhodopsin crystal structureare available from the Protein Data Bank (1HZX) (Teller et al.,Biochemistry 40:7761-72 (2001)). The effects of amino acid substitutionson the structure of rhodopsin, and on the contacts between opsin and11-cis-retinal, or a synthetic retinal, can be determined by molecularmodeling.

The coordinates for the rhodopsin crystal structure from the ProteinData Bank (1HZX) (Teller et al., Biochemistry 40:7761-72 (2001)) can beused to generate a computer model. The addition of hydrogen atoms andoptimization can be done, for example, using Insight II (InsightIIrelease 2000, Accelrys, Inc., San Diego, Calif.). Crystallographic watercan be removed, and water molecules introduced based on the accessiblespace in the extracellular region. Typically, no minimization isperformed before water is added. A water layer (e.g., 5 Å thick) can beused to coat the extracellular part of rhodopsin as well as residues incontact with polar phospholipids heads. All of the water molecules canbe allowed to move freely, as is the extracellular half of rhodopsin,with retinal. If no water cap is put on the cytoplasmic part ofrhodopsin, this part of the molecule can be frozen to preventdegradation of the model.

A water cap can be put on the extracellular part of rhodopsin (togetherwith that part buried in membrane in contact with polar heads ofphospholipids). Water and the extracellular part of rhodopsin can beallowed to move and the movement modeled at any suitable frequency. Forexample, the movement of the modeled rhodopsin can be modeling at 100 pssimulations.

Synthetic retinals can be contacted with an opsin protein underconditions suitable and for a period of time sufficient for theformation of an opsin protein/synthetic retinal complex. The stabilityof the opsin/synthetic retinal complex can be determined by methodsdescribed herein or as known to the skilled artisan. The opsin in theopsin/synthetic retinal complex is stabilized when it exhibits increasedstability (e.g., increased half-life when bound to the synthetic retinalas compared with free opsin (i.e., not bound to retinoid), is lesssensitive to hydroxylamine, exhibits less accumulation in aggresomes, orthe like).

The synthetic retinal can be contacted with the opsin protein in vitroor in vivo. For example, the opsin protein can be synthesized in an invitro translation system (e.g., a wheat germ or reticulocyte lysateexpression system) and the synthetic retinal added to the expressionsystem. The opsin protein can be contacted with the opsin protein exvivo, and then the complex can be administered to a vertebrate eye.

In another aspect, methods of using a synthetic retinal derivative areprovided to restore or stabilize photoreceptor function, or toameliorate photoreceptor loss, in a vertebrate visual system. Asynthetic retinal derivative can be administered to a vertebrate eye(s)having a retinoid deficiency (e.g., a deficiency of 11-cis-retinal), anexcess of free opsin, an excess of retinoid waste (e.g., degradation)products or intermediates in the recycling of all-trans-retinal, or thelike. The vertebrate eye typically comprises a wild-type opsin protein.Methods of determining endogenous retinoid levels in a vertebrate eye,and a deficiency of such retinoids, are disclosed in, for example, U.S.Provisional Patent Application No. 60/538,051 (filed Feb. 12, 2004) (thedisclosure of which is incorporated by reference herein). Other methodsof determining endogenous retinoid levels in a vertebrate eye, and adeficiency of such retinoids, include for example, analysis by highpressure liquid chromatography (HPLC) of retinoids in a sample from asubject. For example, retinoid levels or a deficiency in such levels canbe determined from a blood sample from a subject.

A blood sample can be obtained from a subject and retinoid types andlevels in the sample can be separated and analyzed by normal phase highpressure liquid chromatography (HPLC) (e.g., with a HP1100 HPLC and aBeckman, Ultrasphere-Si, 4.6 mm×250 mm column using 10% ethylacetate/90% hexane at a flow rate of 1.4 ml/minute). The retinoids canbe detected by, for example, detection at 325 nm using a diode-arraydetector and HP Chemstation A.03.03 software. A deficiency in retinoidscan be determined, for example, by comparison of the profile ofretinoids in the sample with a sample from a control subject (e.g., anormal subject).

As used herein, absent, deficient or depleted levels of endogenousretinoid, such as 11-cis-retinal, refer to levels of endogenous retinoidlower than those found in a healthy eye of a vertebrate of the samespecies. A synthetic retinal derivative can spare the requirement forendogenous retinoid.

As used herein, “prophylactic” and “prophylactically” refer to theadministration of a synthetic retinal derivative to preventdeterioration or further deterioration of the vertebrate visual system,as compared with a comparable vertebrate visual system not receiving thesynthetic retinal derivative. The term “restore” refers to a long-term(e.g., as measured in weeks or months) improvement in photoreceptorfunction in a vertebrate visual system, as compared with a comparablevertebrate visual system not receiving the synthetic retinal derivative.The term “stabilize” refers to minimization of additional degradation ina vertebrate visual system, as compared with a comparable vertebratevisual system not receiving the synthetic retinal derivative.

In one aspect, the vertebrate eye is characterized as having LeberCongenital Amaurosis (“LCA”). This disease is a very rare childhoodcondition that effects children from birth or shortly there after. Itaffects both rods and cones in the eye. For example, certain mutationsin the genes encoding RPE65 and LRAT proteins are involved in LCA.Mutations in both genes result in a person's inability to make11-cis-retinal in adequate quantities. Thus, 11-cis-retinal is eitherabsent or present in reduced quantities. In RPE65-defective individuals,retinyl esters build up in the RPE. LRAT-defective individuals areunable to make esters and subsequently secrete any excess retinoids. ForLCA, a synthetic retinal derivative can be used to replace the absent ordepleted 11-cis-retinal.

In another aspect, the vertebrate eye is characterized as havingRetinitis Punctata Albesciens. This disease is a form of RetinitisPigmentosa that exhibits a shortage of 11-cis-retinal in the rods. Asynthetic retinal derivative can be used to replace the absent ordepleted 11-cis retinal.

In another aspect, the vertebrate eye is characterized as havingCongenital Stationary Night Blindness (“CSNB”) or Fundus Albipunctatus.This group of diseases is manifested by night blindness, but there isnot a progressive loss of vision as in the Retinitis Pigmentosa. Someforms of CSNB are due to a delay in the recycling of 11-cis-retinal.Fundus Albipunctatus until recently was thought to be a special case ofCSNB where the retinal appearance is abnormal with hundreds of smallwhite dots appearing in the retina. It has been shown recently that thisis also a progressive disease, although with a much slower progressionthan Retinitis Pigmentosa. It is caused by a gene defect that leads to adelay in the cycling of 11-cis-retinal. Thus, a synthetic retinalderivative(s) can be administered to restore photoreceptor function byretinoid replacement.

In yet another aspect, the vertebrate eye is characterized as havingage-related macular degeneration (“AMD”). AMD can be wet or dry forms.In AMD, vision loss occurs when complications late in the disease eithercause new blood vessels to grow under the retina or the retinaatrophies. Without intending to be bound by any particular theory,excessive production of waste products from the photoreceptors mayoverload the RPE. This is due to a shortfall of 11-cis-retinal availableto bind opsin. Free opsin is not a stable compound and can spontaneouslycause firing of the biochemical reactions of the visual cascade withoutthe addition of light.

Administration of a synthetic retinal derivative to the vertebrate eyecan reduce the deficiency of 11-cis-retinal and quench spontaneousmisfiring of the opsin. Administration of a synthetic retinal derivativecan lessen the production of waste products and/or lessen drusenformation, and reduce or slow vision loss (e.g., choroidalneovascularization and/or chorioretinal atrophy).

In yet other aspects, a synthetic retinal derivative is administered toan aging subject, such as a human. As used herein, an aging humansubject is typically at least 45, or at least 50, or at least 60, or atleast 65 years old. The subject has an aging eye, which is characterizedas having a decrease in night vision and/or contrast sensitivity. Excessunbound opsin randomly excites the visual transduction system. Thiscreates noise in the system and thus more light and more contrast arenecessary to see well. Quenching these free opsin molecules with asynthetic retinal will reduce spontaneous misfiring and increase thesignal to noise ratio, thereby improving night vision and contrastsensitivity.

Synthetic retinal derivatives can be administered to human or othernon-human vertebrates. The synthetic retinal derivative can besubstantially pure, in that it contains less than about 5% or less thanabout 1%, or less than about 0.1%, of other retinoids. A combination ofsynthetic retinal derivatives can be administered.

Synthetic retinal derivatives can be delivered to the eye by anysuitable means, including, for example, oral, intravenous, intramuscularor local administration. Modes of local administration can include, forexample, eye drops, intraocular injection or periocular injection.Periocular injection typically involves injection of the syntheticretinal derivative into the conjunctiva or to the tennon (the fibroustissue overlying the eye). Intraocular injection typically involvesinjection of the synthetic retinal derivative into the vitreous. Theadministration can be non-invasive, such as by eye drops or oral dosageform.

Synthetic retinal derivatives can be formulated, for example, aspharmaceutical compositions for local administration to the eye and/orfor intravenous, intramuscular or oral administration. In someembodiments, the pharmaceutical composition is not a topicalformulation. In other embodiments, the pharmaceutical composition is nota cosmetic formulation.

Synthetic retinal derivatives can be formulated for administration usingpharmaceutically acceptable vehicles as well as techniques routinelyused in the art. A vehicle can be selected according to the solubilityof the synthetic retinal derivative. Suitable pharmaceuticalcompositions include those that are administrable locally to the eye,such as by eye drops, injection or the like. In the case of eye drops,the formulation can also optionally include, for example,ophthalmologically compatible agents such as isotonizing agents such assodium chloride, concentrated glycerin, and the like; buffering agentssuch as sodium phosphate, sodium acetate, and the like; surfactants suchas polyoxyethylene sorbitan mono-oleate (also referred to as Polysorbate80), polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil, andthe like; stabilization agents such as sodium citrate, sodium edentate,and the like; preservatives such as benzalkonium chloride, parabens, andthe like; and other ingredients. Preservatives can be employed, forexample, at a level of from about 0.001 to about 1.0% weight/volume. ThepH of the formulation is usually within the range acceptable toophthalmologic formulations, such as within the range of about pH 4 to8.

Suitable pharmaceutical compositions also include those formulated forinjection. For example, the synthetic retinal derivative can be providedin an injection grade saline solution, in the form of an injectableliposome solution, or other carriers or vehicles. Intraocular andperiocular injections are known to those skilled in the art and aredescribed in numerous publications including, for example, OphthalmicSurgery: Principles of Practice, Ed., G. L. Spaeth, W. B. Sanders Co.,Philadelphia, Pa., U.S.A., pages 85-87 (1990).

A synthetic retinal derivative also can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. The synthetic retinal derivative can be prepared with acarrier(s) that will protect the compound against rapid release, such asa controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, polylactic acid andpolylactic, polyglycolic copolymers (PLG). Many methods for thepreparation of such formulations are known to those skilled in the art.

Suitable oral dosage forms include, for example, tablets, pills,sachets, or capsules of hard or soft gelatin, methylcellulose or ofanother suitable material easily dissolved in the digestive tract.Suitable nontoxic solid carriers can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like. (See, e.g., Remington “PharmaceuticalSciences”, 17 Ed., Gennaro (ed.), Mack Publishing Co., Easton, Pa.(1985).)

The doses of the synthetic retinal derivatives can be suitably selecteddepending on the clinical status, condition and age of the subject,dosage form and the like. In the case of eye drops, a synthetic retinalderivative can be administered, for example, from about 0.01 mg, about0.1 mg, or about 1 mg, to about 25 mg, to about 50 mg, or to about 90 mgper single dose. Eye drops can be administered one or more times perday, as needed. In the case of injections, suitable doses can be, forexample, about 0.0001 mg, about 0.001 mg, about 0.01 mg, or about 0.1 mgto about 10 mg, to about 25 mg, to about 50 mg, or to about 500 mg ofthe synthetic retinal derivative, one to four times per week. In otherembodiments, about 1.0 to about 300 mg of synthetic retinal derivativecan be administered one to three to five times per week.

Oral doses can typically range from about 1.0 to about 1000 mg, one tofour times, or more, per day. An exemplary dosing range for oraladministration is from about 10 to about 250 mg one to three times perday.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

EXAMPLES Example 1

9-cis-retinyl ester restores visual pigment in an LCA mouse model.LRAT−/− mice were gavaged with all-trans-retinyl palmitate,all-trans-retinyl acetate or 9-cis-retinyl acetate as indicated in thelegend to FIG. 1. Following treatment, retinoids were extracted from theeye and liver, and analyzed by HPLC. As shown in FIG. 1 left, treatmentof mice only with 9-cis-retinyl acetate, but not with all-trans-retinylanalogs restored the presence of syn-9-cis-retinal oxime, indicatingformation of the chromophore and restoration vision in these mice. Nosignificant retention of retinoids was observed in liver, where LRAT ishighly expressed, indicating low or no toxicity by retinoids in thisanimal model of human LCA. 9-cis-retinyl ester restores visual pigmentin approximately 5 hr (FIG. 2), while excess of retinoid is removed andmetabolized (as illustrated for 9-cis-retinol).

Example 2

Vitamin A and its derivatives can isomerized upon exposure to light. Forexample, Rao et al. (Tetrahedron Letters 31:3441-44 (1990)) showedphotoisomerization of all-trans-retinol acetate (a derivative of VitaminA) using a broad wavelength UV light could produce a mixture ofall-trans, 13-cis, and 9-cis retinol acetate isomers. However, thismethods is generally inefficient and produces small amounts of suchretinoid.

Methods

Solutions of all-trans-retinoids are made to concentrations of 1 mg/mLin methanol. The solutions are added to a glass petri dish and subjectedto 365 nm UV light using a Bio-Rad GS Genelinker with the stock bulbsreplaced with 8 watt F8T5 bulbs, for varying lengths of time dependenton the target retinoid. This wavelength is beneficial, as shorter wavelength light quickly destroys retinoids. Following UV-treatment, thesolutions are dried down, dissolved in hexane, and purified using normalphase HPLC. Conversion yields vary for each all-trans derivative.Nonisomerized all-trans derivatives, or 13-cis and 11-cis derivativescan be reused in subsequent repetitions, thereby increasing yields.

Results

Production of 9-Cis-Retinyl Acetate from all-Trans-Retinyl Acetate.

All-trans-retinyl acetate (Sigma # R4632) was dissolved in methanol to aconcentration of 1 mg/mL. The solution was poured into a glass petridish and irradiated with 365 nm UV light, inducing isomerization (FIG.3). Two minutes of irradiation yields a mix of isomers, ˜25%9-cis-retinyl acetate, as shown by HPLC (FIG. 4).

The following diagram illustrates some other compounds that can be madewith this method.

R is hydrogen or lower alkyls ranging from C₁ to C₆. R′ is R or anyhigher alkyls such as palmitate, oleate, or complex groups such assuccinate, fumarate, and other functional groups.

Example 3

Levels of 9-cis-RAL oximes (measured as syn- and anti-9-cis-retinylaldehyde) in the eyes of Lrat−/− mice after a single dose or multipledoses of 9-cis-retinyl-acetate (9-cis-R-Ac). Doses of 9-cis-R-Ac wereadministered to Lrat−/− mice by oral gavage in vegetable oil (100%canola oil) in a volume of 500 μl (2.5 mg/ml). The mice weighed about30-50 g. After 3 days, 9-cis-RAL oximes levels were determined by HPLC.Briefly, all experimental procedures related to extraction,derivatization, and separation of retinoids from dissected mouse eyeswere carried out as described previously. See Van Hooser et al., J.Biol. Chem. 277:19173-182 (2002); Van Hooser et al., Proc. Natl. Acad.Sci. USA 97:8623-28 (2000); Maeda et al., J. Neurochem. 85:944-56(2003). All reactions involving retinoids were carried out under dim redlight.

Referring to FIG. 5 a, the level of 9-cis-RAL in Lrat−/− in mouse eyesafter a varying dose of 9-cis-R-Ac is shown. Peaks were identified byretention time and UV spectra and compared to standards. The spikearound 19 min resulted from changes in the solvent composition. Retinoidanalysis was performed on an HP1100 HPLC equipped with a diode arraydetector and HP Chemstation (A.07.01) software, allowing identificationof retinoid isomers according to their specific retention time andabsorption maxima. A normal-phase column (Beckman Ultrasphere Si 5μ, 4.6mm×250 mm) and an isocratic solvent system of 0.5% ethyl acetate inhexane (v/v) for 15 min, followed by 4% ethyl acetate in hexane for 60min at a flow rate of 1.4 ml/min (total 80 min), with detection at 325nm allowed the partial separation of 11-cis-retinyl esters,13-cis-retinyl esters, and all-trans-retinyl esters at 20° C.

Levels of 9-cis-RAL per eye leveled off at doses of about 4-6 μmole.Referring to FIG. 5 b, the level of 9-cis-RAL in Lrat−/− mouse eyesafter a varying size and number of doses of 9-cis-R-Ac is shown. Levelsof 9-cis-RAL accumulated over time. The levels of 9-cis-RAL increasedfrom about 50 mole per eye to about 600 mmole per eye. The gray solidline represents a maximal level of isorhodopsin as measured by the levelof 9-cis-retinal oximes in Lrat−/− mouse eyes after 10 gavages; dashedgray lines indicate the standard deviations. The maximal level ofisorhodopsin is comparable to the level of rhodopsin in wildtype (WT)mice.

Example 4

Levels of chromophore (opsin/retinal complexes) were measured in theeyes of mice after dosing with all-trans-retinoid isoforms or9-cis-retinyl succinate. All-trans-retinyl-palmitate, all-trans-retinylacetate, all-trans-retinal (vitamin A aldehyde), all-trans-retinol(vitamin A), all-trans-retinyl succinate and 9-cis-retinyl succinatewere administered to Lrat−/− mice by oral gavage. Five milligrams of theretinoid isoforms or 9-cis-retinyl succinate were administered in 100%canola oil at a concentration of 40 mg/ml. After 3 days, chromophorelevels (as all-trans-retinal oximes or 9-cis-retinal oximes) weredetermined as described previously. See Van Hooser et al., J. Biol.Chem. 277:19173-182 (2002); Van Hooser et al., Proc. Natl. Acad. Sci.USA 97:8623-28 (2000); Maeda et al., J. Neurochem. 85:944-56 (2003). Allreactions involving retinoids were carried out under dim red light.

Referring to FIG. 6, the all-trans retinoid isoforms had essentially noeffect on restoration of chromophore levels. In contrast, administrationof 9-cis-retinyl succinate restored chromophore levels.

Example 5

A comparison of the bioavailability of orally delivered9-cis-retinaldehyde and 9-cis-retinyl acetate in an LRAT −/− model.9-cis-retinaldehyde and 9-cis-retinyl acetate were administered at low(10 μmoles) and high (15 mmoles) doses to LRAT−/− mice. Chromophorelevels (as 9-cis-retinal oximes) were determined as describedpreviously. See Van Hooser et al., J. Biol. Chem. 277:19173-182 (2002);Van Hooser et al., Proc. Natl. Acad. Sci. USA 97:8623-28 (2000); Maedaet al., J. Neurochem. 85:944-56 (2003). All reactions involvingretinoids were carried out under dim red light.

Referring to FIG. 7, at low and high doses, administration of9-cis-retinyl acetate more efficiently restores chromophore levels than9-cis-retinaldehyde. This effect is more pronounced at low (10 μmoles)doses. Because administration of retinoids can lead to toxicity,pro-drugs such as retinyl esters (e.g., 9-cis-retinyl acetate) provide asuitable bioavailable form to restore chromophore levels while reducingrisk associated with retinoid toxicity.

The previous examples are provided to illustrate but not to limit thescope of the claimed inventions. Other variants of the inventions willbe readily apparent to those of ordinary skill in the art andencompassed by the appended claims. All publications, patents, patentapplications and other references cited herein are hereby incorporatedby reference.

1-84. (canceled)
 85. A method of treating Leber Congenital Amaurosis(LCA), comprising: administering an effective amount of a syntheticretinyl ester to the eye of a human subject, wherein LCA in the humansubject is due to a deficiency in endogenous 11-cis retinal in the eyeand wherein when the synthetic retinyl ester is administered to thehuman subject a functional opsin/chromophore complex is formed in theeye, with the proviso that if the synthetic retinyl ester is9-cis-retinyl ester it is not palmitoyl-9-cis-retinyl ester.
 86. Themethod of claim 85, wherein the human subject has a mutation selectedfrom an RPE65 mutation and an LRAT mutation.
 87. The method of claim 85,wherein the synthetic retinyl ester is a 9-cis-retinyl ester or an11-cis-retinyl ester, wherein the ester substituent comprises acarboxylate radical of a O₂ to C₂₂ polycarboxylic acid.
 88. The methodof claim 85, wherein the synthetic retinyl ester is a 9-cis-retinylester or an 11-cis-retinyl ester, wherein the ester substituentcomprises a carboxylate radical of a C₁ to C₁₀ monocarboxylic acid. 89.The method of claim 85, wherein the synthetic retinyl ester is a9-cis-retinyl ester or an 11-cis-retinyl ester of Formula I, II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI.
 90. Themethod of claim 85, wherein the synthetic retinyl ester is a9-cis-retinyl ester that is a 9-cis-retinyl succinate or a 9-cis-retinylester.
 91. The method of claim 85, wherein the synthetic retinyl esteris locally administered by eye drops, intraocular injection orperiocular injection to the human subject.
 92. The method of claim 85,wherein the synthetic retinyl ester is orally administered to the humansubject.
 93. The method of claim 85, wherein synthetic retinyl ester isprophylactically administered to the human subject.
 94. The method ofclaim 85, wherein the synthetic retinyl ester is formulated forophthalmological administration with a pharmaceutical acceptablevehicle.
 95. The method of claim 85, wherein the synthetic retinyl esteris a 9-cis-retinyl ester that is a 9-cis-retinyl succinate or a9-cis-retinyl ester and the 9-cis-retinyl ester is formulated for oraladministration.
 96. A method of treating Retinitis Pigmentosa (RP),comprising: administering an effective amount of a synthetic retinylester to the eye of a human subject, wherein RP in the human subject isdue to a deficiency in endogenous 11-cis retinal in the eye and whereinwhen the synthetic retinyl ester is administered to the human subject afunctional opsin/chromophore complex is formed in the eye, with theproviso that if the synthetic retinyl ester is 11-cis-retinyl ester itis not 11-cis-retinyl acetate or 11-cis-retinyl palmitate.
 97. Themethod of claim 96, wherein the synthetic retinyl ester is a9-cis-retinyl ester or an 11-cis-retinyl ester, wherein the estersubstituent comprises a carboxylate radical of a C₂ to C₂₂polycarboxylic acid.
 98. The method of claim 97, wherein the syntheticretinyl ester is a 9-cis-retinyl ester or an 11-cis-retinyl ester,wherein the ester substituent comprises a carboxylate radical of a C₁ toC₁₀ monocarboxylic acid.
 99. The method of claim 97, wherein thesynthetic retinyl ester is a 9-cis-retinyl ester or an 11-cis-retinylester of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII,XIV, XV or XVI.
 100. The method of claim 97, wherein the syntheticretinyl ester is a 9-cis-retinyl ester that is a 9-cis-retinyl succinateor a 9-cis-retinyl ester and the 9-cis-retinyl ester is formulated fororal administration.
 101. A method of treating a visual disorder of ahuman subject selected from Retinitis Punctata Albesciens, CongenitalStationary Night Blindness, Fundus Albipunctatus, Age-Related MacularDegeneration, loss of night vision and contrast sensitivity, comprising:administering an effective amount of a synthetic retinyl ester to an eyeof the human subject, wherein the visual disorder in the human subjectis due to a deficiency in endogenous 11-cis retinal in the eye.
 102. Themethod of claim 101, wherein the visual disorder is Age-Related MacularDegeneration.
 103. The method of claim 101, wherein the visual disorderis loss of night vision and contrast sensitivity in an ageing subject.104. The method of claim 101, wherein the synthetic retinyl ester is a9-cis-retinyl ester or an 11-cis-retinyl ester, wherein the estersubstituent comprises a carboxylate radical of a C₂ to C₂₂polycarboxylic acid or a carboxylate radical of a C₁ to C₁₀monocarboxylic acid.